Background The levels of the microRNA-processing enzyme DICER and the hypoxia associated marker carbonic anhydrase 9 (CA9) critically influence tumor growth, aggressiveness, and response to therapy. However, their prognostic value in patients treated with definitive chemoradiotherapy (CRT) for anal squamous cell carcinoma (ASCC) remains elusive. Patients and methods DICER and CA9 were scored in a cohort of a total of 95 ASCC patients by multiplex immunofluorescence and next-generation sequencing for DICER on pre-treatment biopsies. They were correlated with patients’ histopathological characteristics and clinical endpoints locoregional failure-free survival (LRFFS), distant metastases-free survival (DMFS), disease-free survival (DFS) and overall survival (OS). Results We observed a significant inverse correlation between DICER and CA9 expression. In contrast, DICER and CA9 expression did not correlate with age, gender, T/N stage, and grading. High levels of DICER protein and mRNA were prognostic for superior LRFFS (p=0.006) and DFS (p=0.014), while elevated levels of CA9 were associated with decreased LRFFS (p=0.008) and decreased DFS (p=0.023), respectively. In multivariate analyses, DICER levels remained significant for LRFFS and DFS (p=0.020, and p=0.033). A combined DICER high and low CA9 variable indicated a subgroup of patients most likely to respond to CRT with improved LRFFS (p=0.002) and DFS (p=0.006). Conclusion These data indicate that elevated pretreatment levels of CA9 and low levels of DICER are correlated with an unfavorable clinical outcome in patients with ASCC treated with definitive CRT. The superior prognostic power of DICER+ CA9 suggests that DICER and hypoxia may have non-redundant roles in these malignancies.

Background & Aims:Immune checkpoint inhibitor (ICI) therapy has significantly improved the treatment of solid tumors such as hepatocellular carcinoma (HCC); however, most patients fail to respond. Here, we examined whether co-administration of the tumor-penetrating internalizing (i)RGD peptide, which selectively increases tumor vascular permeability in a neuropilin-1-dependent manner, enhances intratumoral delivery and therapeutic efficacy of αPD-L1 in mouse models of HCC. Methods:αPD-L1, with or without iRGD, was administered intravenously to mice bearing endogenous HCCs (TGFα/c-myc and diethylnitrosamine [DEN]/carbon tetrachloride [CCl4] models). Tumor growth was monitored by MRI. Immune cell composition and activation were analyzed by flow cytometry. RNA sequencing was performed on whole tumors and isolated intratumoral CD45+ immune cells. Results:While αPD-L1 monotherapy had minimal impact on tumor progression, combination treatment with iRGD significantly improved therapeutic efficacy, resulting in markedly reduced tumor growth (mean difference -198.2%, p <0.0001 in TGFα/c-myc and -88.8%, p = 0.0159 in DEN/CCl4-induced HCC mice) and increased objective response rates from 0 to 33% (90% confidence interval 14.6-58.6) in TGFα/c-myc mice and to 80% (90% confidence interval 39.6-95.8) in DEN/CCl4-induced HCC mice. Flow cytometry revealed reduced PD-1high CD8+ T cells and enhanced expression of activation markers (Ki67, CD44, IFN-γ) in the combination group. RNA sequencing of CD45+ cells and whole-tumor transcriptomes indicated decreased immunosuppression and increased vascular permeability in mice receiving the combination therapy. Immunoblot analysis showed enhanced accumulation of αPD-L1 in tumors following iRGD co-administration. Conclusions:iRGD co-administration significantly improves the therapeutic efficacy of αPD-L1 in HCC mouse models by increasing intratumoral αPD-L1 delivery and more effectively alleviating the immunosuppressive tumor microenvironment. This non-conjugated, systemic approach holds strong translational potential to enhance ICI responses in patients with HCC. Impact and implications:Immune checkpoint inhibitors (ICIs) show limited efficacy in hepatocellular carcinoma (HCC), to which poor intratumoral drug delivery is likely to contribute. This study demonstrates that intravenous co-administration of the tumor-penetrating peptide iRGD with anti-PD-L1 significantly improves antibody distribution and therapeutic response without increasing toxicity in preclinical HCC models. These findings are highly relevant for clinicians and researchers aiming to enhance ICI effectiveness in solid tumors. Given the ongoing clinical evaluation of iRGD, this simple, non-conjugated strategy offers a feasible and rapidly translatable approach to overcome a key limitation of current ICI therapy and improve outcomes for patients with HCC.

Lipoxygenases peroxidise polyunsaturated fatty acids, resulting in oxylipins, which may act pro- or anti-inflammatory. Arachidonate 15-lipoxygenase type B was shown to play a role in the resolution of keratinocyte inflammation and is upregulated in psoriasis. Its murine ortholog, arachidonate 8-lipoxygenase (Alox8), differs in regiospecificity in that it adds molecular oxygen to the 8th and not 15th carbon of arachidonic acid. This study aimed to determine if Alox8 plays a role in the resolution of murine imiquimod-induced psoriasis. Alox8 knockout (KO) mice, which are not commercially available, were generated with a functional KO targeting the enzyme's active site. Untargeted Lipidomics revealed changes in the skin lipidome from both imiquimod-induced psoriasis as well as between wild-type and KO mice. Furthermore, LC-MS/MS revealed a functional KO with reductions in Alox8-specific oxylipins. Lipid peroxidation marker 4-hydroxynonenal was elevated in the epidermis of wild-type mice from imiquimod treatment, however, it was significantly reduced in Alox8 KO mice. Alox8 KO mice exhibited a thickened epidermis, resulting from reduced DNA damage and increased proliferation. Moreover, immune cell infiltration was enhanced in Alox8 KO mice, including a higher abundance of γδ T cells. Elevated cytokine levels of interleukin-17 and -22, accompanied by keratinocyte-produced C-X-C motif chemokine ligand 1, were detected in the skin of Alox8 KO mice. Additionally, cyclooxygenase 2 expression and prostaglandin E2 levels were enhanced in Alox8 KO mice. These data demonstrate an exacerbated and prolonged inflammatory psoriasis phenotype in Alox8 KO mice, implying that Alox8 aids in the resolution of murine psoriasis.

RATIONALE:Only subset of patients with Non-Small Cell Lung Cancer (NSCLC) benefit from immunotherapy and this is partly due to limited understanding of how oncogenic mutations shape the tumor microenvironment (TME). OBJECTIVES:To define how EGFR and KRAS mutations influence the spatial organization and immune composition of the NSCLC TME. METHODS:We conducted 38-marker high-plex immunofluorescence to 197 NSCLC tumors (>2 million cells) stratified by EGFR and KRAS genotypes. Quantitative phenotyping and spatial analyses, including cellular neighborhoods (CN), nearest neighbors (NNs = 5-20), and spatial proximity profiling (25-100 μm), were performed to assess immune architecture and clinical correlations. MEASUREMENTS AND MAIN RESULTS:EGFR- and KRAS-mutant tumors showed higher tumor cell density and reduced immune infiltration compared with wild-type tumors. Both mutation types were associated with depletion of cytotoxic T cells, dendritic cells, and granulocytes, while EGFR-mutant tumors showed enrichment of M2-like tumor-associated macrophages (TAMs). CN-analysis identified 14 spatial clusters, with reduced cytotoxic and helper T cell-rich neighborhoods in mutant tumors. NN-analysis revealed shorter distances between M2-like TAMs in EGFR-mutant tumors and greater immune exclusion in non-mutants. Spatial proximity revealed higher densities of T-regs, TAMs near tumor cells in KRAS-mutant tumors. All spatial metrics correlated significantly with prognosis in Cox proportional hazards models, highlighting immune cell positioning as an important predictor of outcome. CONCLUSIONS:EGFR- and KRAS-mutant tumors remodel the NSCLC immune landscape, creating distinct immunosuppressive and immune-excluded niches. Spatial proteomics reveals prognostic immune architectures that may guide mutation-directed immunotherapy strategies.

Background:The lung tumor microenvironment (TME) plays a crucial role in the progression and metastasis of lung cancer. It consists of various cell types that interact in complex ways to influence tumor behavior. CD45+ cells, as a component of the TME, have complex and multifaceted roles in lung cancer. The balance between the anti-tumor and pro-tumor functions of CD45+ cells can significantly affect lung cancer outcomes. Understanding these roles is essential for developing targeted therapies that harness the beneficial effects of CD45+ cells while mitigating their harmful effects. Methods:We performed single-cell RNA sequencing of sorted CD45+ immune cells from healthy lungs, orthotopic LLC1 tumors, and KrasLA2 (Kras) genetically engineered tumors. Analyses included immune composition, transcriptional programs, differentiation trajectories, metabolic states, and ligand-receptor-based intercellular communication networks. Results:Four major immune compartments, B cells, T cells, NK cells, and macrophages, underwent model-specific remodeling. LLC1 tumors showed B cell expansion and T and NK cell reduction, with inflammatory, stress-response, and NF-κB/TNF-dominant programs. KrasLA2 tumors retained a balanced immune composition but exhibited metabolic rewiring, elevated antigen-presentation signatures, and selective intercellular signaling. Subclustering revealed specialized changes across B cell (resting, mature, pre-Bcr, late pro-B, plasma), T cell (Cd4+, Cd8+, memory, activated, Treg, Th17), NK cell (Fcgr3high, Fcgr3low, Xcl1+), and macrophage (Ace+, Bcr+, Ccr2+, Cd3+, metabolic, MHCII+) subsets. Ligand-receptor analyses highlighted dense inflammatory networks in LLC1 tumors versus metabolically tuned signaling in KrasLA2 tumors. Conclusion:Distinct CD45+ immune landscapes, characterized by inflammatory suppression in LLC1 and metabolic adaptation in KrasLA2 tumors, shape lung tumor biology. This atlas identifies genotype-specific immune vulnerabilities with potential relevance for precision immunotherapy in non-small cell lung cancer.

AIMS:A subset of endothelial cells referred to as immunomodulatory endothelial cells (IMEC) has been proposed to regulate T-cell responses in atherosclerosis and after myocardial infarction. Here, we studied the inflammation-induced emergence of IMEC and characterized their crosstalk with T cells. METHODS AND RESULTS:An in vitro model to study IMEC was characterized using flow cytometry and proteomics. Endothelial cell-specific translatome and single-cell transcriptome data from a murine atherogenesis model and single-cell transcriptome data from human atherosclerotic arteries were used to determine pathophysiological relevance. Immunopeptidomics was performed to detect antigen presentation. T-cell chemotaxis, adhesion, and activation were assessed through flow cytometry and microscopy. IMEC were induced by treating human endothelial cells with interleukin-1β, interferon-γ, and transforming growth factor-β2 and expressed lower levels of classical endothelial cell markers and disrupted VE-cadherin expression accompanied by impaired barrier function. IMEC expressed major histocompatibility complex (MHC) class II, proteins involved in antigen processing and presentation (CD83, CD80, and CD86) and pro-inflammatory cytokines as well as chemokines, including CXCL9. An IMEC-like subpopulation was identified in the lumen of carotid arteries in a mouse model of accelerated atherogenesis as well as in human atheromas. Conditioned medium from IMEC enhanced the migration of peripheral blood mononuclear cells and induced T-cell chemotaxis, which was partially inhibited by antagonizing CXCL9. IMEC exhibited a significant down-regulation of proteins related to glycosaminoglycan degradation, consistent with the key role of the glycocalyx in the establishment of chemokine gradients. Indeed, the accumulation of heparan sulphates in IMEC contributed to the adhesion of T cells. Notably, IMEC that had been exposed to monocyte lysates presented 627 peptide antigens on MHC class II and induced T-cell expansion. CONCLUSION:Our data highlight the role of IMEC as non-professional antigen-presenting cells that potentially contribute to T cell-mediated immune responses in cardiovascular disease.

Abstract Introduction Lung cancer is the leading cause of cancer-related deaths. Although current T cell-targeting immunotherapies have improved patient survival, the efficacy is still low. Today, macrophage-mediated immune escape via “do not eat me” signaling is brought to light. The leukocyte immunoglobulin-like receptors (LILRs) are new targets for cancer immunotherapy aimed at tumor-associated macrophages (TAMs). Methods TAMs from lung cancer patients were FACS-sorted and processed for RNA sequencing. Opal multiplex staining on patient microarrays was used to investigate the cellular expression of LILRB4 in the tumor microenvironment. Peripheral blood mononuclear cells (PBMCs) were differentiated into macrophages and co-cultured with cancer cells to obtain in vitro TAMs. qPCRs and Western blotting were performed to evaluate the expression levels of LILRs on TAMs. The functional effect of LILRB4 knockdown TAMs (KD) on cancer cells was studied by flow cytometry. RNA-seq was conducted to elucidate how LILRB4 regulates phagocytosis in TAMs. Further LILRB4 blockage was applied on the ex vivo model of human precision-cut lung slices (PCLS) and in in vivo lung cancer models. Results A comprehensive analysis of the expression profiles of LILRs revealed that among LILRs, LILRB4 is highly expressed in TAMs. In vitro, TAMs-specific blockage of LILRB4 had no effect on cancer cell proliferation and apoptosis, but significantly increased their phagocytosis. RNA sequencing identified differential expressed genes in LILRB4 KD TAMs, which indicate that silencing of LILRB4 inhibits cell cycle progression of TAMs. This result was confirmed by flow cytometry and correlated with reduced phagocytic activity in the G2/M phase. In addition, blocking LILRB4 ex vivo and in vivo reduced tumor growth and differentially affected the immune cell composition. Conclusion Taken together, our data suggest that LILRB4 may play a role in macrophage-specific immune evasion in lung cancer and is a promising target for lung cancer immunotherapy. Funding Source Institute for Lung Health Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Acute respiratory distress syndrome (ARDS) remains difficult to treat due to its heterogeneous etiology. In this study, a mouse model of intratracheal lipopolysaccharide (LPS) instillation was used to investigate age- and sex-dependent immune responses. After six hours of LPS exposure and one hour of mechanical ventilation, immune cell subtypes as well as mRNA and protein expression were analyzed. Aged mice exhibited a basal pro-inflammatory immune signature already under sham conditions, whereas young mice displayed a more balanced response with parallel pro- and anti-inflammatory mechanisms. Notably, aged female LPS treated mice exhibited an exclusively pro-inflammatory response. UMAP analysis revealed distinct clustering according to age, sex, and treatment, while aged male mice formed isolated clusters. These findings highlight the importance of considering age- and sex-specific therapeutic strategies for ARDS treatment.

The ErbB2 (HER2)-specific CAR-engineered natural killer (NK) cell line NK-92/5.28.z is under investigation in a phase I clinical trial in glioblastoma patients. In preclinical studies, these cells demonstrated potent CAR-mediated cytotoxicity and stimulated endogenous antitumor immunity in immunocompetent animals. Pro-inflammatory cytokines can contribute to the CAR-NK cells’ immunomodulatory activity, but this may be attenuated by immunosuppressive IL-10 that is also produced in substantial amounts by activated NK-92/5.28.z cells. To prevent IL-10 secretion, we modified the CAR-NK cells to express an intracellular anti-IL-10 antibody, which trapped IL-10 within the endoplasmic reticulum. This did not affect proliferation, phenotype, or cytotoxicity of the resulting NK-92/5.28.z/anti-IL10ER cells but strengthened their ability to mediate maturation of co-cultured dendritic cells and prevented M2-polarization of co-cultured macrophages induced by unmodified CAR-NK cells. In a syngeneic murine glioblastoma model, NK-92/5.28.z/anti-IL10ER cells exhibited enhanced antitumor activity and favored a pro-inflammatory tumor microenvironment characterized by reduced infiltration of IL-10-responsive immunosuppressive cell types. Our findings demonstrate that inhibiting IL-10 secretion improves the therapeutic potential of CAR-engineered NK-92 cells, suggesting this approach as a promising avenue for clinical translation.

Tumor-associated macrophages (TAMs) possess both tumor-promoting and tumor-inhibiting roles. Here, we explore TAMs' anti-tumor functions, focusing on the immune responsive gene 1 (IRG1) and its product, itaconate, in lung cancer development. Spatial metabolomics reveals that endogenous itaconate is markedly depleted within lung tumor regions compared with adjacent non-tumor tissue. Single-cell RNA sequencing shows that macrophages are the primary cells expressing IRG1 in human and mouse lung tumors. Both IRG1 knockout and transplantation of IRG1-depleted bone marrow leads to increased lung tumor growth in various mouse lung tumor models. Additionally, 4-octyl itaconate (Octyl Ita) reduces tumor growth in vitro, in vivo, and in ex vivo human tumor precision-cut lung slices. An integrated multi-omics analysis shows that IRG1/itaconate causes a metabolic shift in cancer cell and pro-tumor macrophages, mainly by inhibiting the pentose phosphate pathway (PPP) through targeting glucose-6-phosphate dehydrogenase (G6PD) activity, thereby suppressing cancer cell growth and transforming pro-tumor macrophages into anti-tumor macrophages. Thus, leveraging IRG1/itaconate's tumor-suppressive effects or using Octyl Ita could be a novel lung cancer therapy.

Post-infectious bronchiolitis obliterans (PiBO) is a chronic lung disease that develops after severe lower respiratory infections and leads to persistent inflammation and fibrotic changes in the small airways. In the present study, gene expression analysis was used to identify differentially expressed genes (DEGs) in sputum cells derived from PiBO patients and compare them to healthy controls. Clinical history, lung function parameters, and induced sputum samples were collected from nine patients with PiBO and eight healthy controls. Multiplex immunohistochemistry (mIHC) as well as mRNA sequencing (MACE-Seq) were performed. Evaluation of the biological targets was done by KEGG pathway enrichment analysis. PiBO patients showed significantly reduced lung function parameters, an increased neutrophil count, and an altered macrophage profile in sputum. Transcriptome analysis revealed significant upregulation of the TNFα-dependent NFκB signalling pathway, as well as significant downregulation of the oxidative phosphorylation (OXPHOS). Linear regression analyses and mIHC indicated a shift in macrophage polarisation that may contribute to the dysregulated gene expression. Notably, expression of these DEGs significantly correlated with FEV1 lung function. These findings indicate a central role of macrophages in the immunopathology of PiBO and contribute to our understanding of the molecular mechanisms involved in the disease process.

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression, and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects BC therapy success and remains a major challenge. Although necroptosis, a lytic, and via damage-associated molecular patterns (DAMP) release, highly immunogenic mode of PCD, might overcome apoptosis resistance, there is an urgent need for physiological and translational human models to model necroptosis and PCD resistance in BC. Here, we apply 3D patient-derived, metastatic human mammary organoids (hMOs) to model apoptosis resistance, necroptosis, and inflammatory signaling using single-cell CITE-sequencing, time-lapse live cell brightfield, and immunofluorescent confocal microscopy, as well as biochemical approaches. Smac mimetic-triggered apoptosis could be confirmed in a panel of BC hMOs. Upon inducing apoptosis resistance with caspase inhibition, BC hMOs rapidly undergo necroptosis with profound MLKL phosphorylation. Necroptotic cell death was preceded by prominent transcriptional upregulation of inflammatory cyto- and chemokines, including interferons, that activate natural killer cells. Finally, necroptosis and the expression and release of inflammatory messengers in metastatic BC hMOs could be attenuated upon the inhibition of linear ubiquitination. We describe a novel experimental platform to model PCD, inflammation, and necroptosis that allows therapeutic screening to overcome chemotherapy resistance in patient-derived metastatic BC hMOs. With this platform, we identified necroptosis-induced interferon signaling, suggesting Smac mimetics and necroptosis as a potential immunotherapy strategy against metastatic BC.

Abstract Glioblastoma (GB) is the most aggressive type of brain cancer with a devastating prognosis. Myeloid cells, especially monocytes and macrophages, comprise the majority of immune cells within the GB tumour microenvironment, where they contribute to the development of an immunosuppressive milieu hindering potential anti-tumour immune responses. Type I IFNs usually facilitate a pro-inflammatory shift in monocytes and macrophages and allow for anti-tumour functions. Using RNA sequencing, we observed that type I IFN-stimulated genes (ISGs) are globally downregulated in primary human monocytes, but not microglia, co-cultured with GB cells both upon direct cell–cell contact and upon the exchange of soluble factors. Surprisingly, the reduction of type I IFN responses did not result from changes in interferon-α/β-receptor availability and activation. Instead, we identified GM-CSF as a critical, GB cell-derived factor, which decreased ISG expression in monocytes at extremely low concentrations via induction of the TGF-β signalling pathway. In line, type I IFN response gene expression and GM-CSF activity in human GB biopsies appeared to be mutually exclusive. Specifically, active GM-CSF signalling appeared to inhibit ISG expression in the same cell as well as in neighbouring monocyte-derived macrophages, which resulted in lower T cell recruitment. In conclusion, our data provide evidence for a so far unknown GM-CSF- and TGF-β-dependent type I IFN inhibitory mechanism in GB with potential implications for future therapeutic approaches.

Abstract Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by systemic chronic inflammation that can lead to heterogeneous clinical manifestations, from mild cutaneous disease to multi-organ damage and failure. Understanding how immune cell composition, their activation status, and metabolic changes contribute to disease is important to identify new biomarkers and therapeutic targets. Methods In this study, we combined spectral flow cytometry and CITE-Seq to profile immune cell subsets and metabolic changes at the single-cell level. Peripheral white blood cells from SLE patients, psoriatic arthritis (PsA) patients, and healthy donors were analyzed using a 50-marker panel that evaluated immune subsets, activation and exhaustion markers, including metabolic dependencies by using SCENITH. The same antibody panel was used in CITE-Seq to validate flow cytometric findings and explore additional metabolic and inflammation pathways. High-dimensional computational analysis was used to identify immune subpopulations with specific metabolic signatures that distinguished SLE from PsA and healthy individuals. Results Across 44 immune cell populations, we observed significant immunophenotypic and immunometabolic changes in both diseases. B cell analysis revealed altered frequencies, different translational activity, and metabolic dependence in early developmental and antigen-experienced cell subsets. SCENITH profiling showed distinct metabolic signatures in CD4+ and CD8+ T cells, with more pronounced differences in naive and memory subsets. In fact, in SLE, T cells exhibited strong dependence on OXPHOS and limited sensitivity to glycolytic inhibition. Finally, PsA patients presented increased NKT-cell frequencies, indicating an involvement of lipid antigens. Conclusion Overall, these findings describe several immune and metabolic dysregulations in SLE and PsA patients, which could be used as potential biomarkers for disease progression and therapeutic intervention. Funding Source Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project 321115009 (GRK 2336/3) Topic Categories Basic Autoimmunity (BA)

Abstract Interleukin-38 (IL-38) is a cytokine of the IL-1 cytokine family that promotes the resolution of inflammation. Resolution mechanisms comprise the induction or recovery of immune tolerance that is lacking in various acute and chronic inflammatory pathologies, including Graft-versus-Host Disease (GvHD). The role of IL-38 in the context of immune tolerance, its primary immune cell targets and underlying molecular mechanisms are not defined. In this study, we investigated the impact of IL-38 on human alloreactivity and in a mouse model of acute GvHD. Our data suggests that monocytes differentiating into macrophages are the main cellular target of IL-38. Specifically, IL-38 reduces antigen presentation capacity in differentiating monocytes through an IL-1 family receptor-independent mechanism, which subsequently avoids T-cell activation. In parallel, IL-38 ameliorates inflammation in allogeneic settings in human and murine GvHD models by promoting the expansion of regulatory T-cells. Our findings indicate that IL-38 promotes immune tolerance during alloreactivity by affecting myeloid cells and T-cells.

Chronic histiocytic intervillositis (CHI) is a placental lesion characterized by an inflammatory response, significantly influencing maternal and fetal outcomes. This study aims to develop a comprehensive morphologic atlas detailing the localization of fetal and maternal macrophages within the context of CHI. We employed immunohistochemical and multiplexing techniques to analyze placental samples, identifying expression patterns and spatial distribution of key macrophage markers, including CD68, CD163, CD14, and HLA-DR. The results revealed a marked accumulation of activated macrophages in both the intervillous space and villous stroma, with distinct differences in morphology and immunophenotype of fetal Hofbauer cells versus maternal macrophages. Our findings contribute to a better understanding of the immune landscape in CHI and provide a valuable resource for further research into placental immune dynamics. By establishing this morphologic atlas, we aim to enhance diagnostic and therapeutic strategies for affected pregnancies, thereby improving the diagnostic approach and making it more straightforward to recognize CHI histologically.

The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor, which is endogenously activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). BLT2 is gaining attention as a potential therapeutic target involved in various pathologies including diabetic wound healing, ophthalmic diseases, and colitis. However, validation of BLT2 as drug target requires chemical probes and pharmacological tools which will allow for application in vivo. In this work, we present the discovery of a novel chemical probe T-10430 for BLT2 agonism following a scaffold-hopping approach. T-10430 exhibits high potency, good selectivity profile, promising physicochemical and PK properties and can potentially serve as orally applicable pharmacological tool for validation of BLT2 as drug target. Using T-10430, we demonstrate the beneficial effect of BLT2 activation in mouse model of psoriasis.

Chronic inflammatory diseases are a significant global burden and are associated with dysregulated resolution of inflammation. Therefore, promoting the process of resolution is a promising therapeutic approach. This study presents the potent anti-inflammatory and pro-resolving effects of a natural product-derived compound called C81. Administration of C81 in a therapeutic window resolved inflammation in the murine imiquimod-induced psoriasis model, and reduced microglial infiltration in a laser-induced choroidal neovascularisation model. Investigations into the underlying mechanisms of C81 identified the DYRK1B/STAT3 axis as a new regulator of inflammatory processes in leukocytes. The inhibition of DYRK1B by C81 resulted in attenuated STAT3 phosphorylation. The depletion of STAT3-regulated gene expression led to the inhibition of leukocyte adhesion and migration due to reduced integrin activation, and in addition to the inhibition of the release of pro-inflammatory mediators such as cytokines and eicosanoids. Importantly, the pro-resolving effects of C81 included the cell type-specific induction of apoptosis in neutrophils and a subsequent increase in efferocytosis. In conclusion, we report the DYRK1B/STAT3 axis as a novel and promising therapeutic target for activating the resolution of inflammation.

RATIONALE: Macrophages are critical players in the lung cancer microenvironment, promoting tumor development and progression. Tumor cells modulate microRNAs in immune cells, dampening their antitumor response and reprogramming them to support tumor growth. However, the molecular mechanisms underlying these immune-tumor interactions are not well understood. METHODS: Using a microRNA (miR) array-based approach, we found that miR-193b is strongly induced in macrophages co-cultured with tumor cells. We investigated the functional significance of this microRNA histologically and by flow cytometry using preclinical mouse models of lung cancer lacking miR-193b in the myeloid cell compartment. Furthermore, we inhibited miR-193b expression in human PBMC-derived macrophages by a specific hairpin inhibitor. We used the conditioned media for further functional validation experiments in vitro and human precision-cut lung slices (PCLS) ex vivo.RESULTS:In vivo, we observed reduced tumor burden in both the orthotropic tumor model (miR-193bfl/flLysMCre) and the oncogene-driven model (KRasLA2miR-193bfl/flLysMCre) due to depletion of miR-193b in the myeloid cell compartment. In addition, we found altered immune cell composition and reduced tumor cell proliferation and vascularization in these myeloid cell-specific miR-193b mouse models. Furthermore, in vitro inhibition of miR-193b by a hairpin inhibitor leads to the activation of macrophages from human PBMCs towards a pro-inflammatory phenotype. Conditioned media from macrophages treated with a miR-193b inhibitor significantly reduced tumor cell proliferation and migration. In contrast, inhibition of miR-193b in tumor cells alone does not appear to affect tumor cell behavior. Further treatment of human PCLS with macrophage-conditioned medium reduces tumor cell proliferation when miR-193b is inhibited. Remarkably, the expression of miR-193b correlates negatively with the overall survival of NSCLC patients depending on gender, subtype and stage of the disease. CONCLUSION: In summary, our results have important implications for the development of targeted immunotherapies for lung cancer. Targeting miR-193b in macrophages and related oncogenic pathways could reverse the tumor-promoting effect of these cells in the tumor microenvironment and enhance the anti-tumor immune response.

RATIONALE: Lung cancer is the most common cause of cancer-related deaths. Although immunotherapies directed against T cells have improved patient survival, the efficacy is still low. Today, macrophage-mediated immune escape is brought to light by the upregulation of “don't eat me” signaling. The “don't eat me” signals are the new targets for cancer immunotherapy aimed at tumor-associated macrophages (TAMs). METHODS: TAMs from lung cancer patients were FACS-sorted and processed for RNA sequencing. Opel multiplex staining on microarrays from lung cancer patients was used to investigate the cellular expression of X gene in the tumor microenvironment. Peripheral blood mononuclear cells (PBMCs) were isolated, differentiated into macrophages and co-cultured with cancer cells to obtain in vitro TAMs. qPCRs, Western blotting and immunofluorescence staining were performed to evaluate the expression level of “don't eat me” signals on TAMs. Flow cytometry was used to investigate the effect of X gene knockdown TAMs (KD) on apoptosis, proliferation and phagocytosis of cancer cells. The same parameters were also tested on the ex vivo model of human precision-cut lung slices (PCLS). RNA sequencing was performed to elucidate the mechanism by which X gene regulates phagocytosis in macrophages. RESULTS: A comprehensive analysis of the expression profiles of “don't eat me” signals in different experimental macrophage models revealed that among the “don't eat me” signals, X gene is highly expressed in TAMs. Interestingly, genetic ablation of X gene in in vitro TAMs reduced the expression of tumor-promoting M2 macrophage genes. At the functional level, macrophage-specific silencing of X gene has no effect on cancer cell proliferation and apoptosis. However, phagocytosis of cancer cells increased significantly after macrophage-specific genetic ablation and antibody-mediated blockade of X gene. RNA sequencing revealed differential gene regulation (DEGs) in TAMs_X gene_KD compared to TAMs. Analysis of these DEGs revealed that silencing of X gene inhibits cell cycle progression of macrophages. This result was confirmed by flow cytometry and correlated with reduced phagocytic activity in the G2/M phase. In addition, blocking X gene on PCLS differentially affected immune cell composition and increased phagocytosis. CONCLUSIONS: Taken together, our data suggest that X gene may play a role in macrophage-specific immune evasion in lung cancer and may be a promising target for immunotherapy of lung cancer.